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Closed Access item An invariant lysine residue is involved in catalysis at the 3′-5′ exonuclease active site of eukaryotic-type DNA polymerases1
|Authors:||Vega, Miguel de|
Lázaro, José M.
|Keywords:||ø29 DNA polymerase, 3′-5′ exonuclease, Site-directed mutagenesis|
|Citation:||Journal of Molecular Biology 270(1): 65-78 (1997)|
|Abstract:||A lysine residue, contained in the motif “ Kx2h”, has been invariantly found in the eukaryotic-type (family B) class of DNA-dependent DNA polymerases with a proofreading function. The importance of this lysine has been assessed by site-directed mutagenesis in the corresponding residue (Lys143) of ø29 DNA polymerase. Substitution of this residue either by arginine or isoleucine severely impaired the catalytic efficiency of the 3′-5′ exonuclease activity, giving a characteristic distributive pattern that contrasts with the processive pattern displayed by the wild-type ø29 DNA polymerase. Exonuclease assays carried out in the presence of a DNA trap, together with direct analysis of enzyme/ssDNA interaction, allowed us to conclude that this altered pattern was due to a reduction in the catalytic rate of these mutants, but not to a weakened association with ssDNA. These phenotypes indicate that the lysine residue of motif Kx2h plays an auxiliary role in catalysis of the exonuclease reaction, in very good agreement with recent crystallographic data showing that the lysine homologue of T4 DNA polymerase is indirectly involved in metal binding at the 3′-5′ exonuclease active site. In agreement with a critical role in proofreading, substitution of Lys143 of ø29 DNA polymerase by arginine or isoleucine produced mutator enzymes that displayed a high frequency of misincorporation. Mutants at Lys143 also showed a reduced DNA polymerization capacity, but only when DNA synthesis was coupled to strand-displacement, an intrinsic property of ø29 DNA polymerase that is specifically affected by mutations at residues directly or indirectly involved in metal binding at the 3′-5′ exonuclease active site.|
|Publisher version (URL):||http://dx.doi.org/10.1006/jmbi.1997.1093|
|Appears in Collections:||(CBM) Artículos|
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